The elevation of intracellular free Ca2+ concentration is an essential signal controlling the differentiation and functions of T lymphocytes. The long-term goal of this proposal is to elucidate the molecular and cellular mechanisms that generate and shape Ca2+ signals in T cells. The Ca2+ release activated Ca2+ (CRAC) channel plays a central role in this process. These channels open in response to the depletion of the Ca2+ stored in the endoplasmic reticulum (ER), but the mechanism linking store depletion to channel activation is not well understood. In addition, Ca2+ entering the cell through CRAC channels interacts with mitochondria and Ca2+ pumps in the plasma membrane to create prolonged and complex Ca2+ signals. Recent developments in fluorescent Ca2+ indicators and fluorescence microscopy have made it possible to address these mechanisms at a subcellular level and visualize microscopic sites of CRAC channel activity. We will address functional interactions between CRAC channels and the ER, mitochondria, and pumps using a combination of approaches, including patch-clamp recording, targeted genetically-encoded Ca2+ indicators, and total internal reflection fluorescence microscopy (TIR-FM). We will distinguish between two models of CRAC activation: physical contact with proteins in the ER, and activation via a diffusible messenger released from the ER. We will explore the role of ER fragmentation in the mechanism of CRAC channel inactivation by Ca2+, and the mechanisms for clustering Ca2+ pumps in the plasma membrane and bringing them close to CRAC channels for functional interaction. The significance of this project is two-fold. First, the results will help to clarify fundamental issues related to store-operated channels and calcium signaling in a wide variety of non-excitable cells. Second, the results of these studies may help identify novel targets for the control of the immune response that may be beneficial in treating autoimmune disorders or immunodeficiencies, and they may help explain immune dysfunction resulting from aberrant operation of the Ca signaling machinery. [unreadable] [unreadable]